RESUMO
Background: Immune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies. Methods: Comprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy. Results: LAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq. Conclusions: These results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade.
Assuntos
Melanoma , Receptor de Morte Celular Programada 1 , Humanos , Antígeno CTLA-4 , Receptor de Morte Celular Programada 1/metabolismo , Leucócitos Mononucleares , Imunofenotipagem , Ligantes , Microambiente Tumoral , Fibrinogênio/uso terapêuticoRESUMO
Purpose: Despite high initial response rates with cytoreductive surgery, conventional chemotherapy and the incorporation of biologic agents, ovarian cancer patients often relapse and die from their disease. New approaches are needed to improve patient outcomes. This study was designed to evaluate the antitumor activity of NEO-201 monoclonal antibody (mAb) in preclinical models of ovarian cancer where the NEO-201 target is highly expressed. Experimental Design: Functional analysis of NEO-201 against tumor cell lines was performed by antibody-dependent cellular cytotoxicity (ADCC) assays. Binding of NEO-201 to tumor tissues and cell lines were determined by immunohistochemistry (IHC) and flow cytometry, respectively. Further characterization of the antigen recognized by NEO-201 was performed by mass spectrometry. Ovarian cancer models were used to evaluate the anti-tumor activity of NEO-201 in vivo. NEO-201 at a concentration of 250 g/mouse was injected intraperitoneally (IP) on days 1, 4, and 8. Human PBMCs were injected IP simultaneously as effector cells. Results: Both IHC and flow cytometry revealed that NEO-201 binds prominently to the colon, pancreatic, and mucinous ovarian cancer tissues and cell lines. Immunoprecipitation of the antigen recognized by NEO-201 was performed in human ovarian, colon, and pancreatic cancer cell lines. From these screening, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) and CEACAM6 were identified as the most likely targets of NEO-201. Our results confirmed that NEO-201 binds different types of cancers; the binding is highly selective for the tumor cells without cross reactivity with the surrounding healthy tissue. Functional analysis revealed that NEO-201 mediates ADCC killing against human ovarian and colorectal carcinoma cell lines in vitro. In addition, NEO-201 inhibited tumor growth in the presence of activated human PBMCs in orthotopic mouse models of both primary and metastatic ovarian cancer. Importantly, NEO-201 prolonged survival of tumor-bearing mice. Conclusions: These data suggested that NEO-201 has an antitumor activity against tumor cells expressing its antigen. Targeting an antigen expressed in tumors, but not in normal tissues, allows patient selection for optimal treatment. These findings strongly indicate that NEO-201 warrants clinical testing as both a novel therapeutic and diagnostic agent for treatment of ovarian carcinomas. A first in human clinical trial evaluating NEO-201 in adults with chemo-resistant solid tumors is ongoing at the NIH clinical Center.
RESUMO
Background: Natural killer (NK) cells are essential to innate immunity and participate in cancer immune surveillance. Heterophilic interactions between carcinoembryonic antigen (CEA) on tumor cells and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on NK cells inhibit NK cell cytotoxicity against tumor cells. NEO-201 is a humanized IgG1 monoclonal antibody that recognizes members of CEACAM family, expressed specifically on a variety of human carcinoma cell lines and tumor tissues. This investigation was designed to determine whether the binding of NEO-201 with CEACAM5 on tumor cells can block the CEACAM5/CEACAM1 interaction to restore antitumor cytotoxicity of NK cells. Materials and Methods: In vitro functional assays, using various human tumor cell lines as target cells and NK-92 cells as effectors, were conducted to assess the ability of NEO-201 to block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to enhance the in vitro killing of tumor cells by NK-92. NK-92 cells were used as a model of direct NK killing of tumor cells because they lack antibody-dependent cellular cytotoxicity activity. Results: Expression profiling revealed that various human carcinoma cell lines expressed different levels of CEACAM5+ and NEO-201+ cells. Addition of NEO-201 significantly enhanced NK-92 cell cytotoxicity against highly CEACAM5+/NEO-201+ expressing tumor cells, suggesting that its activity is correlated with the level of CEACAM5+/NEO-201+ expression. Conclusions: These findings demonstrate that NEO-201 can block the interaction between CEACAM5 on tumor cells and CEACAM1 on NK cells to reverse CEACAM1-dependent inhibition of NK cytotoxicity.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígeno Carcinoembrionário/genética , Carcinoma/tratamento farmacológico , Células Matadoras Naturais/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma/genética , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/genética , Humanos , CamundongosRESUMO
BACKGROUND: A major mechanism of action for therapeutic antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC). ALT-803 is an interleukin-15 superagonist complex that enhances ADCC against human carcinoma cells in vitro and exerts an antitumor activity in murine, rat, and human carcinomas in vivo. The authors investigated the ability of ALT-803 to modulate ADCC mediated by the humanized IgG1 monoclonal antibody (mAb) NEO-201 against human carcinoma cells. MATERIALS AND METHODS: ALT-803 modulating activity on ADCC mediated by NEO-201 was evaluated on several NEO-201 ligand-expressing human carcinoma cells. Purified human natural killer (NK) cells from multiple healthy donors were treated with ALT-803 before their use as effectors in ADCC assay. Modulation of NK cell phenotype and cytotoxic function by exposure to ALT-803 was evaluated by flow cytometry and gene expression analysis. RESULTS: ALT-803 significantly enhanced ADCC mediated by NEO-201. ALT-803 also upregulated NK activating receptors, antiapoptotic factors, and factors involved in the NK cytotoxicity, as well as downregulated gene expression of NK inhibiting receptors. CONCLUSIONS: These findings indicate that ALT-803 can enhance ADCC activity mediated by NEO-201, by modulating NK activation and cytotoxicity, suggesting a possible clinical use of ALT-803 in combination with NEO-201 for the treatment of human carcinomas.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma/tratamento farmacológico , Proteínas/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma/imunologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Voluntários Saudáveis , Humanos , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Interleucina-15/agonistas , Interleucina-15/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Proteínas/uso terapêutico , Proteínas Recombinantes de FusãoRESUMO
The complex signaling networks of the tumor microenvironment that facilitate tumor growth and progression toward metastatic disease are becoming a focus of potential therapeutic options. The chemokine IL-8 is overexpressed in multiple cancer types, including triple-negative breast cancer (TNBC), where it promotes the acquisition of mesenchymal features, stemness, resistance to therapies, and the recruitment of immune-suppressive cells to the tumor site. The present study explores the utility of a clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as a potential therapeutic option for TNBC. HuMax-IL8 was shown to revert mesenchymalization in claudin-low TNBC models both in vitro and in vivo as well as to significantly decrease the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) at the tumor site, an effect substantiated when used in combination with docetaxel. In addition, HuMax-IL8 enhanced the susceptibility of claudin-low breast cancer cells to immune-mediated lysis with NK and antigen-specific T cells in vitro. These results demonstrate the multifaceted way in which neutralizing this single chemokine reverts mesenchymalization, decreases recruitment of MDSCs at the tumor site, assists in immune-mediated killing, and forms the rationale for using HuMax-IL8 in combination with chemotherapy or immune-based therapies for the treatment of TNBC.
Assuntos
Claudinas/metabolismo , Interleucina-8/metabolismo , Células Supressoras Mieloides/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular , Quimiocinas , Docetaxel/farmacologia , Tratamento Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Interleucina-8/genética , Camundongos , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/patologia , Transdução de Sinais , Linfócitos T , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente TumoralRESUMO
Mesenchymalization is a cellular and molecular program in which epithelial cells progressively lose their well-differentiated phenotype and adopt mesenchymal characteristics. Tumor mesenchymalization occurs during the progression of cancer to metastatic disease, and is also associated with resistance to multiple therapeutics, including killing by cytotoxic immune cells. Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1, and emerging research has found higher PD-L1 expression in mesenchymalized tumors. Here, the association between TGF-ß1-mediated mesenchymalization and PD-L1 was investigated in non-small cell lung cancer cells (NSCLC). TGF-ß1 was found to upregulate PD-L1 gene transcription in a Smad2-dependent manner, and a positive association between PD-L1 and phosphorylated Smad2 was found in NSCLC tumors. The potential to target these 2 negative immune regulators with a single agent was investigated using M7824, a novel clinical-stage bifunctional agent that targets both PD-L1 and TGF-ß. Treatment of NSCLC cells with M7824 in vitro and in vivo attenuated features of TGF-ß1-mediated mesenchymalization, including mesenchymal marker expression, proliferation suppression, and chemoresistance. These findings demonstrate that upregulation of tumor cell PD-L1 is a novel mechanism of TGF-ß1-induced immunosuppression in NSCLC, and that treatment with M7824 has the potential to simultaneously block both tumor mesenchymalization and PD-L1-dependent immunosuppression.
RESUMO
Molecular factors that define stem cell identity have recently emerged as oncogenic drivers. For instance, brachyury, a key developmental transcriptional factor, is also implicated in carcinogenesis, most notably of chordoma, through mechanisms that remain elusive. Here, we show that brachyury is a crucial regulator of stemness in chordoma and in more common aggressive cancers. Furthermore, this effect of brachyury is mediated by control of synthesis and stability of Yes-associated protein (YAP), a key regulator of tissue growth and homeostasis, providing an unexpected mechanism of control of YAP expression. We further demonstrate that the brachyury-YAP regulatory pathway is associated with tumor aggressiveness. These results elucidate a mechanism of controlling both tumor stemness and aggressiveness through regulatory coupling of two developmental factors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/genética , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Carcinogênese/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Condroma/genética , Condroma/metabolismo , Condroma/patologia , Proteínas Fetais/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Fosfoproteínas/genética , Proteínas com Domínio T/genética , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Tumor growth and progression are the products of complex signaling networks between different cell types within the tumor and its surrounding stroma. In particular, established tumors are known to stimulate an inflammatory reaction via the secretion of cytokines, chemokines, and growth factors that favor the recruitment of a range of infiltrating immune cell populations into the tumor microenvironment. While potentially able to exert tumor control, this inflammatory reaction is typically seized upon by the tumor to promote its own growth and progression towards metastasis. This review focuses on recent advances in understanding how an established tumor can initiate an inflammatory response via the release of pro-inflammatory mediators, such as IL-6 and IL-8, and their roles in cancer metastasis. In particular, the role of the epithelial-mesenchymal transition (EMT), a phenotypic switch observed in carcinomas that promotes progression towards metastasis, is discussed here in relation to cancer inflammation.
Assuntos
Transição Epitelial-Mesenquimal , Inflamação/patologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Citocinas/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen-A2 allele (HLA-A2)-Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer-cell platforms.
Assuntos
Mapeamento de Epitopos , Epitopos de Linfócito T/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Proteínas Repressoras/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is recognized as a relevant process during the progression of carcinomas towards metastatic disease. Epithelial cancer cells undergoing an EMT program may acquire mesenchymal features, motility, invasiveness, and resistance to a variety of anticancer therapeutics. Preventing or reverting the EMT process in carcinomas has the potential to minimize tumor dissemination and the emergence of therapeutic resistance. One of the strategies currently under investigation to target tumor cells undergoing EMT is the generation of a sustained immune response directed against an essential molecular driver of the process. This review focuses on the current development of immune-mediated anticancer interventions aimed at targeting a transcription factor, brachyury, associated with human tumor EMT. Also presented here is a summary of recent studies demonstrating a role for EMT in tumor resistance to immune effector cytotoxicity, and the study of novel strategies aimed at reverting the EMT to be used in combination with immune-mediated anticancer interventions.
Assuntos
Vacinas Anticâncer/imunologia , Transição Epitelial-Mesenquimal , Proteínas Fetais/imunologia , Neoplasias/patologia , Proteínas com Domínio T/imunologia , Animais , Resistencia a Medicamentos Antineoplásicos , Humanos , ImunoterapiaRESUMO
NEO-201 is a novel humanized IgG1 monoclonal antibody that was derived from an immunogenic preparation of tumor-associated antigens from pooled allogeneic colon tumor tissue extracts. It was found to react against a variety of cultured human carcinoma cell lines and was highly reactive against the majority of tumor tissues from many different carcinomas, including colon, pancreatic, stomach, lung, and breast cancers. NEO-201 also exhibited tumor specificity, as the majority of normal tissues were not recognized by this antibody. Functional assays revealed that treatment with NEO-201 is capable of mediating both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against tumor cells. Furthermore, the growth of human pancreatic xenograft tumors in vivo was largely attenuated by treatment with NEO-201 both alone and in combination with human peripheral blood mononuclear cells as an effector cell source for ADCC. In vivo biodistribution studies in human tumor xenograft-bearing mice revealed that NEO-201 preferentially accumulates in the tumor but not organ tissue. Finally, a single-dose toxicity study in non-human primates demonstrated safety and tolerability of NEO-201, as a transient decrease in circulating neutrophils was the only related adverse effect observed. These findings indicate that NEO-201 warrants clinical testing as both a novel diagnostic and therapeutic agent for the treatment of a broad variety of carcinomas.
RESUMO
Controlling the spread of carcinoma cells to distant organs is the foremost challenge in cancer treatment, as metastatic disease is generally resistant to therapy and is ultimately incurable for the majority of patients. The plasticity of tumor cell phenotype, in which the behaviors and functions of individual tumor cells differ markedly depending upon intrinsic and extrinsic factors, is now known to be a central mechanism in cancer progression. Our expanding knowledge of epithelial and mesenchymal phenotypic states in tumor cells, and the dynamic nature of the transitions between these phenotypes has created new opportunities to intervene to better control the behavior of tumor cells. There are now a variety of innovative pharmacological approaches to preferentially target tumor cells that have acquired mesenchymal features, including cytotoxic agents that directly kill these cells, and inhibitors that block or revert the process of mesenchymalization. Furthermore, novel immunological strategies have been developed to engage the immune system in seeking out and destroying mesenchymalized tumor cells. This review highlights the relevance of phenotypic plasticity in tumor biology, and discusses recently developed pharmacological and immunological means of targeting this phenomenon.
Assuntos
Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Progressão da Doença , Transição Epitelial-Mesenquimal/imunologia , Humanos , Metástase Neoplásica , Neoplasias/imunologia , Neoplasias/patologia , FenótipoRESUMO
Interleukin-8 (IL-8, CXCL8) is a pro-inflammatory chemokine produced by various cell types to recruit leukocytes to sites of infection or tissue injury. Acquisition of IL-8 and/or its receptors CXCR1 and CXCR2 are known to be a relatively common occurrence during tumor progression. Emerging research now indicates that paracrine signaling by tumor-derived IL-8 promotes the trafficking of neutrophils and myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment, which have the ability to dampen anti-tumor immune responses. Furthermore, recent studies have also shown that IL-8 produced by the tumor mass can induce tumor cells to undergo the transdifferentiation process epithelial-to-mesenchymal transition (EMT) in which tumor cells shed their epithelial characteristics and acquire mesenchymal characteristics. EMT can increase metastatic dissemination, stemness, and intrinsic resistance, including to killing by cytotoxic immune cells. This review highlights the dual potential roles that the inflammatory cytokine IL-8 plays in promoting tumor resistance by enhancing the immunosuppressive microenvironment and activating EMT, and then discusses the potential for targeting the IL-8/IL-8 receptor axis to combat these various resistance mechanisms.
RESUMO
PURPOSE: The conversion of tumor cells from an epithelial to a mesenchymal-like phenotype, via a process designated as the epithelial-mesenchymal transition (EMT), is known to mediate tumor resistance to a variety of cell death inducers, including cytotoxic effector immune cells. The goal of this study was to identify and potentially repurpose FDA-approved compounds capable of reducing mesenchymal features of human lung carcinoma cells, which could be used in combination with immunotherapies or chemotherapeutic strategies to improve clinical responses. EXPERIMENTAL DESIGN: In the current report, we have utilized a quantitative high-throughput screening (qHTS) of a pharmaceutical collection of more than 2,000 compounds to identify clinically approved drugs capable of augmenting the sensitivity of mesenchymal-like, lung cancer cells to immune- and chemotherapy-mediated lysis, both in vitro and in vivo RESULTS: The estrogen receptor antagonist fulvestrant was shown to reduce mesenchymal features of lung carcinoma cells, resulting in tumor sensitization to the cytotoxic effect of antigen-specific T cells, natural killer (NK) effector cells, and chemotherapy both in vivo and in vitro CONCLUSIONS: To our knowledge, this is the first report defining a potential role for estrogenic signaling in promoting tumor resistance to immune-mediated cytotoxicity and chemotherapy in lung cancer. Our data demonstrate a robust association between the acquisition of mesenchymal attributes, therapeutic resistance of lung carcinoma cells, and the expression of estrogen receptor 1 (ESR1), supporting further investigations on the role of estrogen signaling in lung cancer progression via the induction of EMT. Clin Cancer Res; 22(24); 6204-16. ©2016 AACR.
Assuntos
Estradiol/análogos & derivados , Receptor alfa de Estrogênio/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/imunologia , Estradiol/farmacologia , Fulvestranto , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
A signaling pathway that is frequently deregulated in human carcinomas and has been explored as a therapeutic target involves the activation of the epidermal growth factor receptor (EGFR). Inhibition of EGFR via the small molecule inhibitors erlotinib and gefitinib commonly results in tumor resistance, even in patients with EGFR-mutant tumors that initially show substantial clinical responses. This study was designed to broaden our understanding of the molecular mechanisms of acquired resistance to erlotinib in lung cancer cells bearing wild type or mutated EGFR. We report here that generation of erlotinib-resistant lung cancer cells in vitro resulted in a phenotypic alteration reminiscent of an epithelial-mesenchymal transition (EMT) concomitant with a robust upregulation of the IL-8/IL-8R axis. Our results also demonstrate that upregulation of p38 MAPK signaling is responsible for the enhanced IL-8 secretion in the erlotinib-resistant tumor cells. Blockade of IL-8 signaling effectively reduced mesenchymal features of the resistant cells and also markedly enhanced their susceptibility to erlotinib. These results provide a rationale for the development of new therapeutic approaches involving blockade of IL-8 signaling for the management of acquired resistance to EGFR inhibition in patients with lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Cloridrato de Erlotinib/uso terapêutico , Interleucina-8/metabolismo , Neoplasias Pulmonares/metabolismo , Células A549 , Animais , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Transição Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Fenótipo , Quinazolinas/uso terapêutico , Transdução de Sinais , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Epithelial-mesenchymal transition (EMT) is a molecular and cellular program in which epithelial cells lose their well-differentiated phenotype and adopt mesenchymal characteristics. This process, which occurs naturally during embryogenesis, has also been shown to be associated with cancer progression and with tumor recurrence following conventional therapies. Brachyury is a transcription factor that mediates EMT during development, and is aberrantly expressed in various human cancers where it promotes tumor cell EMT, metastatic dissemination, and resistance to conventional therapies. We have recently shown that very high expression of brachyury can protect tumor cells against immune cell-mediated cytotoxicity. In seeking to elucidate mechanisms of immunotherapy resistance, we have discovered a novel positive association between brachyury and mucin-1 (MUC1). MUC1 is overexpressed in the majority of carcinomas, and it has been shown to mediate oncogenic signaling and confer resistance to genotoxic agents. We found that MUC1 is concomitantly upregulated in tumor cell lines that highly express brachyury due to an enhancement of MUC1 mRNA stability. Analysis of patient lung tumor tissues also identified a positive association between these two proteins in the majority of samples. Inhibition of MUC1 by siRNA-based gene silencing markedly enhanced the susceptibility of brachyury-expressing cancer cells to killing by tumor necrosis-related apoptosis-inducing ligand (TRAIL) and to perforin/granzyme-dependent lysis by immune cytotoxic cells. These studies confirm a protective role for MUC1 in brachyury-expressing cancer cells, and suggest that inhibition of MUC1 can restore the susceptibility of mesenchymal-like cancer cells to immune attack.
RESUMO
Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.
Assuntos
Adenoviridae/genética , Vacinas contra Adenovirus/uso terapêutico , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Imunoterapia , Neoplasias/terapia , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/imunologia , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/imunologia , Animais , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gramicidin A (GA) is a channel-forming ionophore that renders biological membranes permeable to specific cations which disrupts cellular ionic homeostasis. It is a well-known antibiotic, however it's potential as a therapeutic agent for cancer has not been widely evaluated. In two recently published studies, we showed that GA treatment is toxic to cell lines and tumor xenografts derived from renal cell carcinoma (RCC), a devastating disease that is highly resistant to conventional therapy. GA was found to possess the qualities of both a cytotoxic drug and a targeted angiogenesis inhibitor, and this combination significantly compromised RCC growth in vitro and in vivo. In this review, we summarize our recent research on GA, discuss the possible mechanisms whereby it exerts its anti-tumor effects, and share our perspectives on the future opportunities and challenges to the use of GA as a new anticancer agent.
RESUMO
The Wilms' tumor transcription factor (WT1) was originally classified as a tumor suppressor, but it is now known to also be associated with cancer progression and poor prognosis in several malignancies. WT1 plays an essential role in orchestrating a developmental process known as mesenchymal-to-epithelial transition (MET) during kidney development, but also induces the reverse process, epithelial-to-mesenchymal transition (EMT) during heart development. WT1 is not expressed in the adult kidney, but shows elevated expression in clear cell renal cell carcinoma (ccRCC). However, the role of WT1 in this disease has not been characterized. In this study, we demonstrate that WT1 is upregulated in ccRCC cells that are deficient in the expression of the von Hippel-Lindau tumor suppressor protein (VHL). We found that WT1 transcriptionally activated Snail, a master transcriptional repressor that is known to induce EMT. Although Snail represses E-cadherin and induces mesenchymal characteristics, we found partial maintenance of E-cadherin and associated epithelial characteristics in kidney cells and ccRCC cells that express WT1, since WT1 upregulates E-cadherin expression and competes with Snail repression. These findings support a novel paradigm in which WT1 induces an epithelial-mesenchymal hybrid transition (EMHT), characterized by Snail up-regulation with E-cadherin maintenance, a tumor cell differentiation state in which cancer cells keep both EMT and MET characteristics which may promote tumor cell plasticity and tumor progression.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Transição Epitelial-Mesenquimal/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas Wnt/genética , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteínas WT1/genéticaRESUMO
Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane potential by permeabilizing membranes to specific ions. Gramicidin A is a channel-forming ionophore that forms a hydrophilic membrane pore that permits the rapid passage of monovalent cations. Previously, we found that gramicidin A induces cellular energy stress and cell death in renal cell carcinoma (RCC) cell lines. RCC is a therapy-resistant cancer that is characterized by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). Here, we demonstrate that gramicidin A inhibits HIF in RCC cells. We found that gramicidin A destabilized HIF-1α and HIF-2α proteins in both normoxic and hypoxic conditions, which in turn diminished HIF transcriptional activity and the expression of various hypoxia-response genes. Mechanistic examination revealed that gramicidin A accelerates O(2)-dependent downregulation of HIF by upregulating the expression of the von Hippel-Lindau (VHL) tumor suppressor protein, which targets hydroxylated HIF for proteasomal degradation. Furthermore, gramicidin A reduced the growth of human RCC xenograft tumors without causing significant toxicity in mice. Gramicidin A-treated tumors also displayed physiologic and molecular features consistent with the inhibition of HIF-dependent angiogenesis. Taken together, these results demonstrate a new role for gramicidin A as a potent inhibitor of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC.